Membrane-Cytoskeletal Remodeling in Platelet Biogenesis

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 DESCRIPTION (provided by applicant): Project Summary Defects in platelet production and function play substantial roles in cardiovascular, bleeding, and inflammatory diseases. More complete understanding of platelet biogenesis will yield insights and advances in human health. My mentor's lab discovered a new cytoskeletal component, Cdc42-interacting protein 4 (CIP4), in a yeast two-hybrid screen with the Src kinase Lyn as bait. CIP4 is a BAR protein that coordinates membrane and cytoskeletal remodeling. Through its SH3 domain, CIP4 interacts with Wiskott-Aldrich Syndrome Protein (WASP) or dynamin. Wiskott-Aldrich Syndrome is characterized by thrombocytopenia. My lab generated CIP4-knockout (KO) mice that displayed thrombocytopenia. I determined that the mechanism for thrombocytopenia in CIP4-KO mouse megakaryocytes involves decreased proplatelet formation and reduced demarcation membrane system, which are not observed in WASP-KO mouse megakaryocytes. In addition, unlike WASP-KO mice, CIP4 KO mice show decreased platelet-microparticle release into the plasma. I hypothesize that CIP4-dependent thrombocytopenia involves dynamin. Surprisingly, cells with dynamin knockdown showed increased microparticle release. Since dynamin's chief role is to promote endocytic vesicle formation, this variation of membrane scission mediated by dynamin would be novel. The goal of my proposed research is to determine the mechanism by which CIP4-dynamin pathway regulates membrane remodeling in normal platelet biogenesis and how this may impact platelet biogenesis. I hypothesize that loss of dynamin, affects membrane intracellular trafficking in megakaryocytes, resulting in more membrane being available for demarcation membrane system invagination and for microparticle release. To address this hypothesis, I propose the following two specific aims: 1) establish that loss of dynamin promotes formation of the demarcation membrane system and 2) define the thrombogenic potential from microparticles produced by megakaryocytes/platelets with reduced dynamin activity. This proposal combines cell biology, biochemistry, biophysics, and advanced imaging with animal modeling to establish a new pathway of membrane remodeling in platelet biogenesis and thrombogenesis. This K08 award provides me with intensive laboratory training, mentorship, and committee oversight so that I can successfully develop into an independent physician- scientist. Research and training plan will be carried at Northwestern University under the mentorships of Drs. Seth Corey and Susan Quaggin with graduate school coursework in cell biology and advice from a committee of well-established investigators in platelet biogenesis (Joseph Italiano), thrombogenesis (Xiaoping Du) and hematopoiesis (Liz Eklund).